Polarization-resolved nonlinear optical microscopy can provide label-free images of anisotropic biomaterials. Existing techniques are often limited by their complexity, chromaticity, or slowness. In this article, we propose a simple implementation based on the passive splitting of the excitation beam into slightly delayed orthogonally polarized pulse trains. Label-free second- and third-harmonic signals corresponding to each polarization state are separated by time-resolved photon counting, resulting in the acquisition of image pairs that are automatically registered with submicron spatial precision and nanosecond simultaneity. We demonstrate the utility of this small-footprint approach for live studies by presenting structural imaging of tubulin, myelinated axons, and blood vessels in zebrafish larvae, dynamic imaging of flowing microcrystals in the zebrafish larval otolith cavity, and mitotic spindles in dividing embryonic cells.

中文翻译：

---

用于探测动态生物过程的多重正交偏振谐波成像


偏振分辨非线性光学显微镜可以提供各向异性生物材料的无标记图像。现有技术通常受到其复杂性、色度或缓慢性的限制。在本文中，我们提出了一种基于将激励光束无源分裂为稍微延迟的正交偏振脉冲序列的简单实现。通过时间分辨光子计数来分离对应于每个偏振态的无标记二次和三次谐波信号，从而采集以亚微米空间精度和纳秒同步性自动配准的图像对。我们通过展示斑马鱼幼虫中微管蛋白、有髓轴突和血管的结构成像、斑马鱼幼虫耳石腔中流动微晶的动态成像以及分裂胚胎细胞中的有丝分裂纺锤体，展示了这种小足迹方法在实时研究中的实用性。